The Saupe matrix describing protein alignment in a liquid crystalline medium contains five independent elements, enabling the generation of up to five linearly independent alignment conditions. Measurement of internuclear residual dipolar couplings (RDCs) by NMR spectroscopy under these conditions, orthogonal in five-dimensional alignment space, provides access to the amplitude, asymmetry, and direction of motions of the internuclear vector. We previously demonstrated for the small protein domain GB3 (56 residues) that suitably orthogonal alignment conditions can be generated in a single liquid crystalline medium of Pf1 phage, by generating a series of conservative mutants that have negligible impact on the time-averaged backbone structure of the domain. Mutations involve changes in the charge of several solvent-exposed sidechains, as well as extension of the protein by either an N- or C-terminal His-tag peptide, commonly used for protein purification. These protein mutants map out the five-dimensional alignment space, providing unique insights into the structure and dynamics, and providing access to anisotropic parameters such as the 13C, 15N and 1H chemical shielding tensors. In collaboration with Michael F. Summers (HHMI, U. of Maryland BC) we are working on the development of techniques applicable to the structure of large retroviral RNA structures, and to identify the structure-function relationship to viral packaging.